The present invention relates generally to an improved microdroplet test apparatus used in determining human leukocyte antigens (HLA antigens) by measuring lymphocyte cytotoxicity. More specifically, the present invention relates to an improved microdroplet test tray design and improved tray cover structures which are designed to enhance the accuracy and usefulness of the microdroplet test system.
The microdroplet lymphocyte cytotoxicity test was introduced in 1964 by Terasaki, P. I., McClelland, J. D.: Microdroplet Assay of Human Serum Cytotoxins. Nature 204: 998-1000, 1964. Since that time, the microdroplet lymphocyte cytotoxicity test has gained universal acceptance as the method of choice to test for HLA antigens.
The basic microlymphocyte cytotoxicity test consists of reacting 0.001 milliliters of lymphocytes with 0.001 milliliters of a specific antibody which is known to be reactive with a specific HLA antigen. If the lymphocytes contain the specific antigen being tested for, the antibodies will bind to the lymphocytes. Measurement of the degree of binding between the specific antibody and the lymphocytes is accomplished by adding 0.005 milliliters of rabbit complement into the test well containing the lymphocytes and antibody. The rabbit complement promotes lysing of those lymphocytes which have reacted with the antibody. Measurement of the antibody-lymphocyte reaction is then measured by viewing the test solution in the test cell with a 10.times. microscope to determine the degree of lymphocyte lysis.
In order to provide a suitable means for handling these minute quantities of reagents and additionally to provide an apparatus in which numerous tests can be carried out simultaneously, a plastic tray with multiple microtest wells was developed. These trays, which are commonly known as "Terasaki Trays" are widely known and used for microdroplet testing of HLA antigens. The microtest wells in the Terasaki tray are circular frusto-conical wells having an inwardly sloping funnel-shaped straight side wall of constant slope. The inwardly sloping side wall terminates at the microtest well bottom. The microtest well bottom has a circular cross section which is designed to be equal to the area which is visible in the single field of a 10.times. microscope objective when the test wells are viewed from directly above and adjacent to the tray. This allows the technician to view the entire test reaction (i.e. cell lysis) in one field of the microscope.
Although the Terasaki tray has experienced wide popularity and is well suited for its intended purpose, some difficulty has been experienced with lymphocytes sticking or not flowing completely down the constant slope microtest well side wall. It is critical that the entire sample of lymphocytes to be tested be placed at the bottom of the microtest well. If the lymphocytes become stuck or otherwise adhere to the upper portions of the microtest cell side wall, a false positive test result is possible. It therefore would be desirable to provide an improved tray in which the test wells are shaped to prevent retention of lymphocytes on the microtest cell side walls to thereby promote localization of the lymphocytes at or near the microtest well bottom.
During HLA testing, one or more reagents which are themselves colored or which produce a colored product may be utilized. Further, certain test solutions may be opaque. These colored test solutions make it difficult to count the number of lysed leukocytes remaining at the bottom of the microtest well after completion of the test. It would be desirable to provide an apparatus in which the amount of colored solution through which the technician must look to view the leukocytes can be reduced.
The microtest tray can also be used for various testing procedures in which a colored product is produced. In these situations, it is many times desirable to increase the depth of test solution in the microtest well without increasing the total amount of reagents and cells utilized in order to increase the detection limits of the procedure. It would be desirable to provide some type of apparatus which could be combined with the Terasaki Tray to provide this desired increase in optical path through the test solution in order to maximize color intensity for measurement.